Method for increasing luminescence assay sensitivity

ABSTRACT

The invention provides kits and methods for increasing the sensitivity of a bio-luminescent assay, which employ an organic compound that, for instance, reduces luminescence that is not dependent on the presence of an analyte by at least about 10 fold and reduces luminescence that is dependent on the presence of an analyte by less than about 7 fold, reduces luminescence generated by luminogenic molecules not bound to an enzyme by at least about 10 fold and reduces the luminescence generated by luminogenic molecules bound to an enzyme by less than about 7 fold, or reduces autoluminescence by at least about 10 fold and reduces luminescence that is dependent on the presence of an analyte by less than about 7 fold.

FIELD OF THE INVENTION

The present invention relates generally to the fields of cell biologyand molecular biology. In particular, this invention relates to methods,compositions and kits for increasing the sensitivity of a luminescenceassay measurement.

BACKGROUND OF THE INVENTION

Reporter molecules are routinely used to monitor molecular events in thefields of biology, biochemistry, immunology, cell biology and molecularbiology. For example, reporter molecules are employed in assays wherethe levels of the reporter molecule are due to transcription from aspecific promoter linked to the reporter molecule. These assays can beused to study eukaryotic gene expression, receptor activity,transcription factors, intracellular signaling, mRNA processing, proteinfolding, and the like. Reporter molecules that are typically used insuch assays include radioactive isotopes, fluorescent agents, enzymes,and luminescent agents. See for example, Akhavan-Tafti, et al, in:Bioluminescence and Chemiluminescence. Fundamentals and Applied Aspects.Proceedings of the 8th International Symposium on Bioluminescence andChemiluminescence. Cambridge, September 1994. Eds. Campbel, Kricka,Stanley. John Wiley and Sons 1994.

Two luminescent enzymes that are particularly useful in assay systemsare firefly luciferase and Renilla reniformis luciferase. The substratesfor these luciferases and the reaction products they produce are shownin FIGS. 1 and 2. The quantity of light (i.e. the number of photons)produced in the reaction, can be measured and used to calculate theconcentration of luminescent enzyme in the reaction.

Firefly luciferase is a 61 kDa monomeric protein that does not requirepost-translational processing for enzymatic activity. Thus, it functionsas a genetic reporter immediately upon translation. Photon emission isachieved through oxidation of beetle luciferin in a reaction thatrequires ATP, Mg²⁺ and O₂ (FIG. 1).

Renilla luciferase is a 36 kDa monomeric protein that is composed of 3%carbohydrate when purified from its natural source, Renilla reniformis.Like firefly luciferase, post-translational modification of Renillaluciferase is not required for its activity, and it functions as agenetic reporter immediately following translation. The luminescentreaction catalyzed by Renilla luciferase utilizes O₂ andcoelenterate-luciferin, also called coelenterazine (FIG. 2).

Luminescent reactions can be used to detect very small quantities of aparticular analyte, the substance being identified and measured in ananalysis. For example, luminescent reactions can be used to detect andquantify proteases, lipases, phosphatases, peroxidases, glycosidases,and various metabolites such as ATP or NADH. Luminescent reactions canalso be used to detect and quantify analytes through bindinginteractions, such as those mediated by antibodies and nucleotideprobes. Typically, luminescent reactions can be used to detect less than1×10⁻¹⁶ moles of analyte in a sample, often less than 1×10⁻¹⁹ moles. Inluminescence, commonly detected analytes are the luciferases, especiallyfirefly luciferase and Renilla luciferase. Most often these analytes areused to quantify phenomena associated with their creation through geneexpression and protein synthesis. Other luminescent enzymes used asanalytes include, but are not limited to, aequorin, Vargula luciferase,and other marine luciferases.

When using luminescence to measure an analyte, it is preferred thatlittle or no light is produced by reactions that are not dependent onthe presence of the analyte. This is the case with firefly luciferase.Under typical firefly luciferase assay conditions, luminescence cannotbe detected when the firefly luciferase is not present. In contrast toassays employing firefly luciferase, light can generally be detected inRenilla luciferase assay systems when the Renilla luciferase is notpresent. Luminescence that is not dependent on the catalytic activity ofa luminescent enzyme is termed autoluminescence. For example,autoluminescence can be caused by spontaneous oxidation of theluminogenic substrate coelenterazine.

Luminescence that is not dependent on the on the presence of an analyte(e.g. autoluminescence) can limit the usefulness of an analytical assayby reducing the ability to accurately measure the quantity of lightresulting from the activity of the analyte. In particular, thesensitivity of luminescent assays containing coelenterazine or itsstructural analogs is reduced due to autoluminescence. Additionally, theaddition of various components to the assay system, such as lipids(especially above the critical micelle concentration or CMC),hydrophobic proteins (especially those with a defined three-dimensionalstructure), and cells or other biological materials containinghydrophobic microenvironments, can greatly increase autoluminescence.

Assay sensitivity may also be reduced by luminescence from an unrelatedluminogenic molecule. The unrelated luminogenic molecule may be presentdue to contamination of the analytical assay, or due to a separateanalytical luminescence assay performed in the same reaction mixture. Ineither case, the sensitivity of an analytical luminescence assay couldbe improved by reducing the luminescence that is not dependent on thepresence of the analyte.

SUMMARY OF THE INVENTION

Applicants have discovered that the sensitivity of luminescence assayscan be improved by carrying out the assay in the presence of one or moreorganic compounds that reduce analyte-independent luminescence. Inparticular, Applicant has unexpectedly discovered that theanalyte-independent luminescence can be reduced without similarlyreducing analyte-dependent luminescence. Preferably, theanalyte-dependent luminescence is reduced by a lower fold than theanalyte-independent luminescence, or the analyte dependent luminescenceremains about the same or increases. Accordingly, the invention providesa method for increasing the sensitivity of a luminescent assaycomprising carrying out the assay in the presence of an organic compoundthat reduces luminescence that is not dependent on the presence of ananalyte by a factor of at least about 10 fold, and that reducesluminescence that is dependent on the presence of an analyte by lessthan about 7 fold.

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that reduces luminescence generated by luminogenicmolecules not bound to an enzyme by at least about 10 fold, and thatreduces the luminescence generated by luminogenic molecules bound to anenzyme by less than about 7 fold.

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that reduces autoluminescence by at least about 10fold, and that reduces luminescence that is dependent on the presence ofan analyte by less than about 7 fold.

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that comprises a selenium atom.

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that comprises a carbon-selenium bond.

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that comprises a carbon selenium double bond (C═Se).

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that comprises a carbon-selenium single bond (C—Se)

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that comprises a carbon-sulfur double bond (C═S).

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that comprises a carbon atom bound to both aselenium atom and a nitrogen atom.

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that comprises a carbon atom bound to both a sulfuratom and a nitrogen atom.

The invention also provides a method for increasing the sensitivity of aluminescent assay comprising carrying out the assay in the presence ofan organic compound that comprises a sulfur atom bound to two carbonatoms, wherein the analyte-independent luminescence is reduced by atleast about 10 fold. Preferably, the analyte-dependent luminescence isreduced by less than 7 fold.

The invention also provides an assay kit comprising packaging materialcontaining 1) a luminogenic substrate of a luminescent enzyme, or aluminogenic enzyme; and 2) an organic compound for increasing thesensitivity of a luminescent assay. Preferably, the organic molecule iscapable of 1) reducing the luminescence that is not dependent on thepresence of an analyte by a factor of at least about 10 fold, andreducing the luminescence that is dependent on the presence of ananalyte by less than about 7 fold; 2) reducing the luminescencegenerated by luminogenic molecules not bound to an enzyme by at leastabout 10 fold, and reducing the luminescence generated by luminogenicmolecules bound to an enzyme by less than about 7 fold; or 3) reducingautoluminescence by at least about 10 fold, and reducing luminescencethat is dependent on the presence of an analyte by less than about 7fold.

The invention also provides novel compounds disclosed herein that areuseful to increase the sensitivity of a luminescent assay.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates chemiluminescent reaction catalyzed by fireflyluciferase.

FIG. 2 illustrates chemiluminescent reaction catalyzed by Renillaluciferase.

FIG. 3 illustrates a dioxetane intermediate in the colenterazineautoluminescence pathway.

FIG. 4 shows representative compounds (1–11) that reduce autoluminescence.

DETAILED DESCRIPTION

Before the present invention is disclosed and described in detail, it isto be understood that this invention is not limited to specific assayformats, materials or reagents, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

As used in the specification and the appended claims, the singular forms“a”, “an” and “the” include plural referents unless the context clearlydictates otherwise. In this specification and in the claims that follow,reference will be made to a number of terms that shall be defined tohave the following meanings, unless otherwise described:

The term “halo” as used herein denotes fluoro, chloro, bromo, or iodo.

The terms “Alkyl”, “alkoxy”, “alkenyl”, “alkynyl”, etc. as used hereindenote both branched and unbranched groups; but reference to anindividual radical such as “propyl” embraces only the straight,unbranched chain radical, a branched chain isomer such as “isopropyl”being specifically referred to.

The term “Aryl”, as used herein, denotes a monocyclic or polycyclichydrocarbon radical comprising 6 to 30 atoms wherein at least one ringis aromatic. Preferably, aryl denotes a phenyl radical or an ortho-fusedbicyclic carbocyclic radical having about nine to ten ring atoms inwhich at least one ring is aromatic. “Heteroaryl” encompasses a radicalof a monocyclic aromatic ring containing five or six ring atomsconsisting of carbon and one to four heteroatoms each selected from thegroup consisting of non-peroxide oxygen, sulfur, and N(X) wherein X isabsent or is H, O, (C₁–C₄)alkyl, phenyl or benzyl, as well as a radicalof a polycyclic ring comprising 8 to 30 atoms derived therefrom.Preferably, heteroaryl encompasses a radical attached via a ring carbonof a monocyclic aromatic ring containing five or six ring atomsconsisting of carbon and one to four heteroatoms each selected from thegroup consisting of non-peroxide oxygen, sulfur, and N(X) wherein X isabsent or is H, O, (C₁–C₄)alkyl, phenyl or benzyl, as well as a radicalof an ortho-fused bicyclic heterocycle of about eight to ten ring atomsderived therefrom, particularly a benz-derivative or one derived byfusing a propylene, trimethylene, or tetramethylene diradical thereto.

The term “analyte”, as used herein is a substance to be detected in atest sample. In luminescence assays, commonly detected analytes includethe luciferases, especially firefly luciferase and Renilla luciferase.Other luminescent enzymes used as analytes include, but are not limitedto, aequorin, Vargula luciferase, and other marine luciferases.Additionally, luminescent reactions can be used to detect and quantifyanalytes such as proteases, lipases, phosphatases, peroxidases,glycosidases, and various metabolites such as ATP or NADH. Luminescentreactions can also be used to detect and quantify analytes throughbinding interactions, such as those mediated by antibodies andnucleotide probes. In certain cases, analyte-dependent luminescence canbe coupled to the activity of a luminescent enzyme. For example,alkaline phosphatase (AP) could be detected by using a phosphoderivative of luciferin. By this strategy, luciferin is generated by theaction of AP, which then yields light by reaction with luciferase. Theinstant invention would allow the AP assay to be run after a separatehorseradish peroxidase/luminol reaction. With respect to the analyte AP,a compound as described herein, could be added to reduce theanalyte-independent luminescence caused by horseradish peroxidase.

The term “autoluminesence” as used herein, refers to the release oflight from a luminogenic molecule that does not result from enzymaticaction on the luminogenic molecule.

The term “increase the sensitivity of a luminescent assay” as usedherein means increasing the precision of the assay or improving theability to measure the presence of a small amount of an analyte with theassay. For example, the sensitivity of a luminescent assay can beincreased by reducing analyte-independent luminescence. Preferably,analyte-independent luminescence is reduced, and a minimal reduction, noreduction, or an increase in analyte-dependent luminescence results.Additionally, analyte-independent luminesence is preferably reduced by agreater fold than analyte-dependent luminescence.

The term “luminescent,” as used herein, includes bio-luminescence (i.elight produced by a living organism), chemi-luminescence (light producedwhen a chemical reaction proceeds), and electrochemical-luminescence.When the enzyme involved has evolved in an organism by natural selectionfor the purpose of generating light, or the enzyme involved is a mutatedderivative of such an enzyme, the luminescent reactions are also called“bioluminescent reactions” and the enzyme involved is also called a“bioluminescent enzyme.” Examples are firefly luciferase, Renillaluciferase, Cypridina luciferase, Aequorin photoprotein, Obelinphotoprotein, and the like.

The term “luminescent assay” or “luminescence assay” includes any assaythat generates light based on the presence of an analyte. Such assaysinclude assays that employ one or more luciferase enzymes (e.g. fireflyluciferase, Renilla luciferase, Cypridina luciferase, and the like).

The term “luminogenic enzyme,” as used herein includes enzymes thatcatalyze a reaction that produces light, or that lead to the productionof light. For example, the term includes firefly luciferase, Renillaluciferase, Cypridina luciferase, Aequorin photoprotein, Obelinphotoprotein, and the like.

The term “luminogenic molecule” as used herein refers to a moleculecapable of creating light via a chemical reaction (e.g. luciferin,coelenterazine, or a functional analog thereof). Generally, aluminogenic molecule is either a high energy molecular species (e.g. astabilized dioxetane), or it is transformed into a high energy molecularspecies by a chemical reaction. The chemical reaction is usuallyoxidation by oxygen, superoxide, or peroxide. In each case, the energywithin the luminogenic molecule is released by the chemical reaction.Although at least some of this energy is released as photons of light,the energy can also be released in other forms, such as heat. Theluminogenic molecules that do not yield light disperse their energythrough alternative modes, often termed “dark pathways”.

The term “luminogenic molecule not bound to an enzyme” as used herein,includes a luminogenic molecule that is not bound to an enzyme (e.g.firefly luciferase, Renilla luciferase, Cypridina luciferase, and thelike) that catalyzes a reaction that produces light.

The term “luminogenic molecules bound to an enzyme” as used hereinincludes a luminogenic molecule that is bound to an enzyme thatcatalyzes a reaction that produces light.

The term “luminescence that is dependent on the presence of an analyte,”or “analyte-dependent luminescence” as used herein, includesluminescence that results from a chemical reaction that involves ananalyte, as well as luminescence that correlates with the presence of ananalyte either directly or indirectly.

The term “luminescence that is not dependent on the presence of ananalyte,” or “analyte-independent luminescence” as used herein, includesluminescence resulting from autoluminescence of a luminogenic substrateas well as luminescence resulting from an unrelated luminogenic moleculepresent in an assay mixture.

The term “quench” as used herein means to reduce the yield of photonsfrom a luminescent reaction. The term includes preventing an analytefrom being detected or being detectable, and may occur either directlyor indirectly. Agents that can be used to quench a reaction are known as“quenching agents.”

Applicant has discovered that it is possible to increase the sensitivityof a luminescent assay by carrying out the assay in the presence of anorganic compound that reduces analyte-independent luminescence. Thisfinding is unexpected. Using procedures similar to those describedherein, one skilled in the art can identify compounds that are suitablefor increasing the sensitivity of a luminescent assay. The structure ofthe compound is not critical provided the compound is capable ofincreasing the sensitivity of a luminescent assay.

In particular, applicant has discovered that compounds that comprise asulfur atom or a selenium atom are particularly useful for increasingthe sensitivity of a luminescent assay. The remaining chemical structureof the compound that comprises a selenium atom or a sulfur atom is notcritical, provided the structure does not interfere with the function ofthe compound. Preferred compounds have low toxicity at concentrationsused in the invention, and can be stored, transported, and disposed ofinexpensively.

Suitable compounds include organic compounds (i.e. compounds thatcomprise one or more carbon atoms). Suitable organic compounds cancomprise a carbon-sulfur bond or a carbon-selenium bond, for examplesuitable organic compounds can comprise a carbon-sulfur double bond(C═S), a carbon selenium double bond (C═Se), a carbon-sulfur single bond(C—S), or carbon-selenium single bond (C—Se). Suitable organic compoundscan also comprise a carbon bound mercapto group (C—SH) or a sulfur atombound to two catbon atoms (C—S—C). Preferred compounds are lipophyllicin nature.

Suitable compounds that comprise a carbon sulfur double bond or a carbonselenium double bond include for example compounds of formula (I):

wherein X is S or Se; R₁ and R₂ are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₂–C₂₀)alkenyl,(C₂–C₂₀)alkynyl, aryl, heteroaryl, or NR_(a)R_(b); or R₁ and R₂ togetherwith the carbon to which they are attached form a 5, 6, 7, or 8 memberedsaturated or unsaturated ring comprising carbon and optionallycomprising 1, 2, or 3 heteroatoms selected from oxy (—O—), thio (—S—),or nitrogen (—NR_(c))—, wherein said ring is optionally substituted with1, 2, or 3 halo, hydroxy, oxo, thioxo, carboxy, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl; and R_(a), R_(b) and R_(c) are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkynyl, aryl, heteroaryl; wherein any(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₂–C₂₀)alkenyl(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, or (C₂–C₂₀)alkynyl of R₁, R₂,R_(a), R_(b), and R_(c) is optionally substituted with one or more (e.g1, 2, 3, or 4) halo, hydroxy, mercapto, oxo, thioxo, carboxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; andwherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, hydroxy, mercapto, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl; or a salt thereof.

Suitable compounds that comprise a mercapto group include for examplecompounds of the formula R₃SH wherein: R₃ is (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl; wherein any (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl,(C₂–C₂₀)alkenyl, or (C₂–C₂₀)alkynyl of R₃ is optionally substituted withone or more (e.g 1, 2, 3, or 4) halo, hydroxy, mercapto oxo, thioxo,carboxy, (C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, orNR_(d)R_(e); wherein R_(d) and R_(e) are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl aryl, or heteroaryl; andwherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, mercapto, hydroxy, oxo, carboxy, cyano,nitro, trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl; or a salt thereof.

Other suitable compounds include for example compounds of the formulaR₄NCS wherein: R₄ is (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl,(C₂–C₂₀)alkynyl, aryl, or heteroaryl; wherein any (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, or (C₂–C₂₀)alkynyl of R₃ isoptionally substituted with one or more (e.g 1, 2, 3, or 4) halo,hydroxy, mercapto oxo, thioxo, carboxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, or NR_(f)R_(g); wherein R_(f)and R_(g) are each independently hydrogen, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl aryl, or heteroaryl; and wherein any aryl orheteroaryl is optionally substituted with one or more (1, 2, 3, or 4)halo, mercapto, hydroxy, oxo, carboxy, cyano, nitro, trifluoromethyl,trifluoromethoxy, (C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkanoyloxy, sulfo or(C₁–C₂₀)alkoxycarbonyl; or a salt thereof.

Other suitable compounds that comprise a carbon-selenium single bond ora carbon sulfur single bond include compounds of formula R₅—X—R₆wherein:

X is —S— or —Se—;

R₅ is (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl,(C₂–C₂₀)alkynyl, aryl, or heteroaryl; and R₆ is hydrogen, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl;

or R₅ and R₆ together with X form a heteroaryl;

wherein any (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, or(C₂–C₂₀)alkynyl of R₅ or R₆ is optionally substituted with one or more(e.g 1, 2, 3, or 4) halo, hydroxy, mercapto oxo, thioxo, carboxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, orNR_(k)R_(m);

wherein R_(k) and R_(m) are each independently hydrogen, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl aryl, or heteroaryl; and

wherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, mercapto, hydroxy, oxo, carboxy, cyano,nitro, trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl; or a salt thereof.

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents

Specifically, (C₁–C₂₀)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₃–C₈)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₁–C₂₀)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy,butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;(C₂–C₂₀)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl,2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl,1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl;(C₂–C₂₀)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁–C₂₀)alkanoyl can be acetyl, propanoyl or butanoyl;(C₁–C₂₀)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₂–C₂₀)alkanoyloxy can be acetoxy, propanoyloxy,butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can bephenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl,triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl,pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide),thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or itsN-oxide) or quinolyl (or its N-oxide).

Specifically, R₁ and R₂ can each independently be hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,aryl, heteroaryl, or NR_(a)R_(b); wherein R_(a) and R_(b) are eachindependently hydrogen, (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl,(C₂–C₂₀)alkenyl, (C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl,(C₂–C₂₀)alkynyl, aryl, or heteroaryl; wherein any (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₂–C₂₀)alkenyl (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, or (C₂–C₂₀)alkynyl of R₁, R₂, R_(a), and R_(b)is optionally substituted with 1 or 2 halo, hydroxy, mercapto, oxo,thioxo, carboxy, (C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, orheteroaryl; and wherein any aryl or heteroaryl is optionally substitutedwith one or more halo, hydroxy, mercapto, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl.

Specifically, R₁ and R₂ can each independently be hydrogen,(C₁–C₂₀)alkyl, (C₂–C₁₀)alkenyl, (C₂–C₁₀)alkynyl, aryl, or NR_(a)R_(b).

Specifically, R₁ and R₂ together with the carbon to which they areattached can form a 5 or 6 membered saturated or unsaturated ringcomprising carbon and optionally comprising 1 or 2 heteroatoms selectedfrom oxy (—O—), thio (—S—), or nitrogen (—NR_(c))—, wherein said ring isoptionally substituted with 1, 2, or 3 halo, hydroxy, oxo, thioxo,carboxy, (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkenyl,(C₂–C₂₀)alkynyl, aryl, or heteroaryl; wherein R_(c) is hydrogen,(C₁–C₂₀)alkyl, (C₃–C₂₀)cycloalkyl, (C₂–C₂₀)alkenyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkynyl, aryl, heteroaryl; wherein any(C₁–C₂₀)alkyl, (C₃–C₂₀)cycloalkyl, (C₁–C₂₀)alkoxy, (C₂–C₂₀)alkenyl(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, or (C₂–C₂₀)alkynyl of R₁, R₂,and R_(c) is optionally substituted with one or more halo, hydroxy,mercapto, oxo, thioxo, carboxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; and wherein any aryl orheteroaryl is optionally substituted with one or more halo, hydroxy,mercapto, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl.

Specifically, R₁ and R₂ can each independently be NR_(a)R_(b); whereinR_(a) and R_(b) are each independently hydrogen, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkynyl, aryl, heteroaryl; wherein any(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, or (C₂–C₂₀)alkynyl is optionally substitutedwith one or more halo, hydroxy, mercapto, oxo, thioxo, carboxy, aryl, orheteroaryl; and wherein any aryl or heteroaryl is optionally substitutedwith one or more halo, hydroxy, mercapto, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl.

Specifically, R₁ and R₂ can each independently be amino, (C₁–C₂₀)alkyl,(C₁–C₂₀)alkylamino, allylamino, 2-hydroxyethylamino, phenylamino, or4-thiazoylamino.

Specifically, R₁ and R₂ can each independently be amino, methyl,allylamino, 2-hydroxyethylamino, phenylamino, or 4-thiazoylamino.

A specific value for R₃ is (C₁–C₂₀)alkyl optionally substituted with oneor more halo, mercapto oxo, thioxo, carboxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, or NR_(d)R_(e).

A specific value for R₃ is 2-aminoethyl, 2-amino-2-carboxyethyl, or2-acylamino-2-carboxyethyl.

A specific value for R₄ is aryl, optionally substituted with one or morehalo, mercapto, hydroxy, oxo, carboxy, cyano, nitro, trifluoromethyl,trifluoromethoxy, (C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkanoyloxy, sulfo or(C₁–C₂₀)alkoxycarbonyl.

Specifically, R₅ is (C₁–C₁₀)alkyl, (C₃–C₆)cycloalkyl, (C₂–C₁₀)alkenyl,(C₂–C₁₀)alkynyl, aryl, or heteroaryl; and R₆ is hydrogen, (C₁–C₁₀)alkyl,(C₃–C₆)cycloalkyl, (C₂–C₁₀)alkenyl, (C₂–C₁₀)alkynyl, aryl, orheteroaryl.

Specifically, R₅ and R₆ together with X form a heteroaryl.

Preferred organic compounds exclude polypeptides and proteins comprisingone or more mercapto (C—SH) groups.

Preferred organic compounds exclude compounds that comprise one or moremercapto (C—SH) groups.

A preferred organic compound is a compound of formula 1–11 as shown inFIG. 4. A more preferred compound is thiourea.

The compounds described hereinabove are available from commercialsources or can be prepared from commercially available startingmaterials using procedures that are known in the field of syntheticchemistry. For example, see Jerry March, Advanced Organic Chemistry, 4thed. Wiley-Interscience, John Wiley and Sons, New York, 1992.

In cases where compounds are sufficiently basic or acidic to form stablesalts, use of the compounds as salts in the methods of the invention maybe appropriate. Examples of suitable salts include organic acid additionsalts, for example, tosylate, methanesulfonate, acetate, citrate,malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate,and α-glycerophosphate salts. Suitable inorganic salts may also beformed, including hydrochloride, sulfate, nitrate, bicarbonate, andcarbonate salts.

Salts can be obtained using standard procedures well known in the art,for example by reacting a sufficiently basic compound with a suitableacid. Alkali metal (for example, sodium, potassium or lithium) oralkaline earth metal (for example calcium) salts can also be used.

When used in accord with the methods of the invention, the compoundsdescribed herein can be present in a luminescence reaction at anyconcentration that increases the sensitivity of the assay. The optimumconcentration of a given compound will depend on the luminescentreagent(s) employed, and on the specific conditions under which a givenassay is carried out. However, suitable concentrations can be determinedusing standard techniques that are available in the art.

Specifically, the compound that can increase the sensitivity of theassay can be present in a luminescence reaction at a concentration of atleast about 0.1 μM, or at a concentration of at least about 0.1 mM. Morespecifically, the compound can be present in the luminescence reactionat a concentration in the range from about 0.1 μM to about 500 mM(inclusive), or in the range from about 1 μM to about 250 mM(inclusive). Preferably, the compound is present at a concentration inthe range from about 10 μM to about 100 mM (inclusive).

Specifically, the assay can be performed in the presence of whole cells.

Specifically, the assay can be carried out in a solvent comprising atleast about 10% water. More specifically, the invention can be carriedout in a solvent comprising at least about 25% water, or at least about40% water.

Preferably, in the practice of the methods of the invention, theanalyte-independent luminescence is reduced by at least about 10 fold,or more preferably by at least about 20 fold, at least about 50 fold, orat least about 100 fold in the present of a compound, while theanalyte-dependent luminescence is reduced by less than about 7 fold,about 5 fold, about 3 fold, or about 2 fold. For example, a relativelight unit value of 5 produced in the presence of the compound while arelative light unit value of 100 produced in the absence of the compoundreflects a decrease in luminescence in the presence of the compound by20 fold.

Preferably in the practice of the methods of the invention, theluminescence generated by luminogenic molecules not bound to an enzymeis reduced by at least about 10 fold, or more preferably by at leastabout 20 fold, at least about 50 fold, or at least about 100 fold, whilethe luminescence generated by luminogenic molecules bound to an enzymeis reduced by less than about 7 fold, about 5 fold, about 3 fold, orabout 2 fold. The luminescence generated by luminonogenic moleculesbound to an enzyme is preferably reduced by a lower fold than the folddecrease in luminescence generated by luminogenic molecules not bound toan enzyme.

Preferably in the practice of the methods of the invention,autoluminescence is reduced by at least about 10 fold, or morepreferably by at least about 20 fold, at least about 50 fold, or atleast about 100 fold, while luminescence that is dependent on thepresence of an analyte is reduced by less than about 7 fold, about 5fold, about 3 fold, or about 2 fold. The luminescence that is dependenton the presence of an analyte is preferably reduced by a lower fold thanthe fold decrease in autoluminescence.

Preferably in the practice of the methods of the invention, when anassay is carried out in the presence of a compound that comprises asulfur atom or a selenium atom, analyte-independent luminescence isreduced by at least about 10 fold, or more preferably by at least about20 fold, at least about 50 fold, or at least about 100 fold.

Preferably in the practice of the methods of the invention, when anassay is carried out in the presence of a compound that comprises asulfur atom or a selenium atom, analyte-dependent luminescence isreduced by less than about 7 fold, about 5 fold, about 3 fold, or about2 fold.

For kits of the invention the enzyme substrate, enzyme, and the compoundcan each be contained in a separate container, or they can be containedin a single container. The kit can optionally comprise a buffer solutionsuitable for use in a luminescent assay, and the enzyme substrate orenzyme, and the buffer solution can optionally be contained in a singlecontainer. Additionally, the compound and the buffer solution canoptionally be contained in a single container. The kits can alsooptionally comprise a second substrate (e.g. a substrate for fireflyluciferase or Renilla luciferase), or a quenching agent for aluminescent enzyme reaction. The kits can also optionally comprise ATP,or can optionally comprise both a luminogenic substrate of a luminescentenzyme, and a luminogenic enzyme.

The ability of a compound to increase the sensitivity of a luminescentassay can be determined using assays that are well known to the art, orusing the assays described in the Examples herein below.

Compounds identified herein have been shown to be useful for increasingthe sensitivity of luminescent assays. The compounds are particularlyuseful for reducing luminescence that results from the decomposition ofintermediate dioxetane rings. Thus, in addition to being useful forincreasing the sensitivity of luminescent assays (e.g. bioluminescent,chemiluminescent, or electroluminescent assays), the compounds are alsouseful for reducing luminescence in other systems that involveintermediate dioxetane rings and the like.

The invention will now be illustrated by the following non-limitingExamples. Compounds 1–11 (FIG. 4) are readily available from commercialsources.

EXAMPLE 1 Improved Assay Sensitivity

Experiments to assess the ability of representative organic compounds(“compounds”) to increase luminescence assay sensitivity were performedunder the conditions described below in Format A and Format B. Improvedluminescence assay sensitivity is demonstrated by the ability of thecompounds to decrease the analyte-independent luminescence resultingfrom the oxidation of coelenterazine, while causing lesser or minimalreduction to the analyte-dependent luminescence, i.e. coelenterazine inthe presence of Renilla luciferase. It is demonstrated herein that thecompound causes a lower-fold decrease in luminescence when the analyteof interest is present than the fold decrease in luminescence when theanalyte of interest is absent. The enzymatic luminescence measurementmay have an autoluminescence component.

In fact, for the majority of experiments described herein, an increasein the enzymatic luminescence measurement was observed when the compoundbeing tested was present. Because autoluminescence is typically verylow, in order to observe a more pronounced effect of the compounds onautoluminescence, autoluminescence was enhanced by adding a detergent,increasing the pH, adding hydrogen peroxide, adding DMSO, adding BSA, oradding sodium hydrosulfite.

Format A Assays were performed in the presence of Steady Glo® reagent(SG) and Stop & Glo® reagent (S+G), (Promega Corporation, Madison Wis.).A total reaction volume of 150 μl consisted of:

50 μl F-12 (Ham) media+1 mg/ml gelatin (with or without enzyme¹)

50 μl S+G (containing substrate²)

50 μl SG

-   -   ¹ Renilla luciferase enzyme was added to F-12 cell culture media        containing 1% gelatin, to a concentration of approximately        2.5ng/50 μl media. Reactions in the absence of Renilla        luciferase reveal the effect of the compound on autoluminescence        while reactions in the presence of enzyme reveal the effect of        the compound on Renilla luciferase-catalyzed luminescence.    -   ² The S+G reagent was prepared as per manufacturer's        instructions, with the exception that for these experiments a        S+G solvent three times more concentrated than normal was used        to resuspend the S+G substrate. Under these conditions, a higher        concentration of coelenterazine in the S+G was needed for        substrate to reach saturation conditions.

The compound to be tested was re-suspended in either SG or S+G reagentto a final concentration of SG or S+G of (1×). The compound was added sothe final concentration in the 150 μl total volume would be that listedin Table 1 and the reagent was diluted to the a final of 50 μl withwater. For controls, the SG or S+G reagent was brought up to 50 μl withwater or with the solvent used to dissolve the compound of interest. Forexample, if a compound needed to be dissolved in DMSO (dimethylsulphoxide), an equal volume of DMSO was added to the control reaction.Unless otherwise indicated, the compounds were first dissolved in water.The same result can be obtained by adding the compound to be testeddirectly to the media portion of the reaction instead of to the SG orS+G.

For each concentration of a particular compound, a mixture containingall of the components in sufficient amounts for four reactions (i.e. 200μl media, 200 μl SG, 200 μl S+G) was assembled. From this mixture, 150μl was dispensed into triplicate wells on a 96-well plate.Alternatively, reactions were sometimes assembled in each well of theplate by adding each of the 50 μl portions and mixing. The plate wasincubated at 22° C. and after 5 minutes the luminescence was measuredusing a Dynex plate luminometer (1 second measurement per well).

Format B Experiments were performed in a reaction volume of 150 μlMatthew's Buffer (referred to herein as MB) as either a standard MBcomposition or a modified MB composition as described below. As withFormat A, reactions with and without Renilla luciferase were carried outto observe the effect of the organic compounds on assay sensitivity. Inorder to be able to add the reaction components such as enzyme,substrate, detergent, and compound to be tested; the reaction wasassembled in 3 portions as follows:

50 μl MB (with or without enzyme³) 50 μl MB with Coelenterazine (with orwithout detergent⁴) 50 μl MB (with or without compound⁵) ³Enzyme wasadded to 1X MB to a concentration of approximately 2.5 ng/50 μl buffer⁴Detergents are known to increase the level of autoluminescence. Forcompleteness, the effect of the compounds on autoliminescence and onenzymatic luminescence was evaluated in the presence and absence ofdetergent. ⁵2X MB was used to make 1X MB with the compound to be testedat various concentrations and water. For controls, 1X MB was made withonly water or with water and the addition of the solvent used tosolubilize the compound to be tested. For example, if the compound to betested was dissolved in DMSO, an equal amount of only DMSO was added tothe control MB sample.1× Matthew's buffer standard composition consists of:

100 mM potassium phosphate

500 mM sodium chloride

1 mM ethylenediaminetetraacetic acid (EDTA)

0.1 mg/ml bovine serum albumin (BSA)

pH 7.4

The BSA functions as an enzyme stabilizer and, in the standard MBcomposition, enhances coelenterazine autoluminescence but not to theextent of the autoluminescence generated when detergent is present. Inorder to observe the autoluminescence enhanced only by the detergent,for the majority of the experiments, BSA was replaced with porcinegelatin as the enzyme stabilizer at a final concentration of 0.15 mg/mlor 0.45 mg/ml. Taking all the variants into account the format can besub-divided in 4 different versions:

B1 BSA/Detergent B2 BSA/No Detergent B3 Gelatin/Detergent B4 Gelatin/NoDetergent

Reactions were carried out in triplicate by adding each of the 50 μlportions to microtiter plate wells and mixing. The resulting relativelight units generated per well was measured immediately using a DynexMLX Microtiter plate luminometer or a Wallac 1450 MicroBeta Trilux plateluminometer (1 second/well) or alternatively, the plate was incubated at22° C. and read after 5 minutes in the same fashion.

Results in Table 1 herein below are shown as:

a) fold-decrease in non-enzymatic autoluminescence measurement in thepresence of the compound when compared to the absence of the compoundand,

b) effect of the compound on enzymatic luminescence measurement in thepresence of the compound when compared to control samples lacking onlythe compound.

For example, a result of “decreased 7.4 fold” indicates that theluminescence measurement in the presence of the compound was 7.4 timesless than the luminescence measurement in the absence of the compound.In all cases, the fold decrease in luminescence not associated with thepresence of Renilla luciferase (autoluminescence) was greater than thefold decrease in luminescence associated with the presence of Renillaluciferase. Thus, the compounds reduce the luminescence not associatedwith enzymatic activity of the analyte to a greater degree than theluminescence associated with the enzymatic activity of the analyte.

TABLE 1 See FIG. 4 for compound identity. Format overview: AMedia:Steady Glo:Stop & Glo B1 Matthew's Buffer with BSA and detergentB2 Matthew's Buffer with BSA without detergent B3 Matthew's Buffer withgelatin and detergent B4 Matthew's Buffer with gelatin without detergentmM Fold compound decrease Com- (in final auto- Effect on pound soln)Format luminescence luminescence 1 33 A 15 No effect 1 316 B2 2Increased 1.2 fold 1 10 B4 2 Increased 2.1 fold 1 100 B3 290 No effect 1100 B4 8.5 Increased 1.4 fold 1 50 B3 + 500 Increased 5 fold 17% DMSO 150 B4 + 15 Increased 7 fold 17% DMSO 1 3 B3 + 21.8 Decreased 7.4 fold 10mM sodium hydrosulfate 1 3 B4 + 2.6 No effect 10 mM sodium hydrosulfate1 3 B3 120 No effect 1 3 B3 with 100 No effect Tween-20 1 3 B3 with 6Increased 1.6 fold Zwittergent 4 10 A 3 No effect 4 30 B3 500 Decreased4.6 fold 4 30 B4 3.9 Increased 1.2 fold 5 10 A 115 Decreased 10 fold 632 A + 65 Decreased 1.5 fold 17% DMSO 6 32 B3 + 660 Increased 1.6 fold33% DMSO 6 32 B4 + 120 Increased 4.7 fold 33% DMSO 2 10 A 100 No effect2 10 B3 70 No effect 3 33 A 100 Increased 1.2 fold 3 33 B3 545 Increased1.2 fold 3 33 B4 3.4 Increased 2.3 fold K SCN 10 B3 55 No effect K SCN10 B4 1.7 Increased 1.2 fold 7 30 B3 7 Increased 1.2 fold 7 30 B4 6.2Increased 2.9 fold 8 100 A 6 No effect 8 30 B3 20 No effect 8 30 B4 3.5Increased 1.5 fold 9 30 B3 13 Decreased 1.4 fold 9 30 B4 2.5 No effect 9100 B4 3 Increased 4.7 fold 11 30 B3 2 No effect 11 30 B4 2.3 Increased1.5 fold 11 100 B4 3.7 Increased 1.8 fold

EXAMPLE 2 Reduction of Autoluminescence Generated by Other Substrates

In addition to looking at the effect of representative compounds onautoluminescence generated from native coelenterazine (PromegaCorporation, Madison Wis.) the effect of representative compounds onautoluminescence generated by other substrates was investigated.Coelenterazine analogs N, F, H, HPC, and CP were obtained from MolecularProbes, Eugene Oreg. Cypridina luciferin was obtained from NanoLightTechnology, Pittsburgh, Pa. Beetle luciferin was obtained from PromegaCorporation, Madison Wis. In order to see a more pronounced effect ofthe compounds, autoluminescence was enhanced as described in Example 1by the addition of DMSO or detergent (1% Tergitol NP-9/1% Antifoam®) inMatthew's Buffer. Autoluminescence was also enhanced by addition of H₂O₂or by raising the pH of the reaction containing native coelenterazine.Experimental conditions are grouped under Format C, sub-divided asfollows:

C1 Alternative Substrates in DMSO

-   -   100 μl per well consisting of:    -   94 μl DMSO    -   1 μl substrate at 3 mM (30 μM final substrate concentration)    -   5 μl of compound dissolved in water or water as the control

C2 Alternative Substrates in MB with Detergent

-   -   100 μl per well consisting of:    -   94 μl MB with gelatin in place of BSA and detergent    -   1 μl substrate at 3 mM (30 μM final substrate concentration)    -   5 μl of compound dissolved in water or water as the control

C3 MB with BSA, pH 9

-   -   100 μl per well consisting of:    -   90 μl MB (standard composition but at pH 9, 30 μM        coelenterazine, no detergent present)    -   5 μl H₂O₂ at 30.7% (1.5% final)    -   5 μl of compound dissolved in water or water as the control

Reactions were carried out in triplicate by adding each of thecomponents to microtiter plate wells and mixing. The light output wasmeasured immediately using a Dynex MLX Microtiter plate luminometer or aWallac 1450 MicroBeta Trilux plate luminometer (1 second/well). Resultsare shown in Table 2.

TABLE 2 mM thiourea Fold decrease Substrate Concentration Formatautoluminescence native 25 C1 4.8 coelenterazine coelenterazine 25 C1 5analog N coelenterazine 25 C1 2.6 analog F coelenterazine 25 C1 1.8analog H coelenterazine 25 C1 7.2 analog HPC coelenterazine 25 C1 8.7analog CP Cypridina 25 C1 4.2 coelenterazine Beetle 25 C1 3.8 luciferinin alkaline environment native 25 C2 1100 coelenterazine coelenterazine25 C2 950 analog N coelenterazine 25 C2 770 analog F coelenterazine 25C2 720 analog H coelenterazine 25 C2 910 analog HPC coelenterazine 25 C2900 analog CP Cypridina 25 C2 310 coelenterazine native 50 C3 48coelenterazine

EXAMPLE 3 Reduction of Luminescence Generated by ChemiluminescentSubstrates CDP-Star® and Luminol

The effect of a representative compound on chemiluminescent reactionscontaining CDP-Star® or Luminol was measured. CDP-Star® is a stabilized1,2-dioxetane chemiluminescent enzyme substrate, a high energyluminogenic molecule, used in the detection of alkaline phosphatase andalkaline phosphatase conjugates in solution and in membrane-basedassays. CDP-Star® was obtained from Tropix PE Biosystems, Bedford, Mass.CDP-Star® substrate produces a light signal when it is activated byalkaline phosphatase. Alkaline phosphatase dephosphorylates thesubstrate, yielding an anion that accumulates due to its long half-life.Since the ultimate light production involves decomposition of the anion,a delay precedes constant signal output, resulting in a glow of lightthat lasts for hours to days. Luminol(5-Amino-2,3-didydro-1,4-phthalazinedione) was obtained from Sigma, St.Louis, Mo. Luminol is a widely used chemiluminescent reagent, thatluminesces upon oxidation. Experimental conditions are grouped underFormat D, sub-divided as follows:

D1 CDP-Star®+Thiourea in Water

In microtiter plate wells, various amounts of CDP-Star® reagent aslisted below were mixed with the representative compound in water (orwater alone as control) and measured in a luminometer as previouslydescribed (1 second/well).

50% 50 μl CDP-Star®+50 μl 0.5 M thiourea (250 mM CDP-Star® final conc.)

75% 75 μl CDP-Star®+25 μl 0.5 M thiourea (125 mM CDP-Star® final conc.)

95% 95 μl CDP-Star®+5 μl 0.5 M thiourea (25 mM CDP-Star® final conc.)

D2 CDP-Star® Undiluted

Thiourea was dissolved directly into CDP-Star® reagent at aconcentration of 10 mM. Control reactions contained CDP-Star® reagentalone (100 μl per well) and light output was measured on a MLXMicrotiter plate luminometer or a Wallac 1450 MicroBeta Trilux plateluminometer (1 second read per well).

Parallel wells containing 0.28 pg alkaline phosphatase were alsomeasured to monitor the CDP-Star's integrity and activity in theseconditions.

D3 Luminol

When a solution containing Luminol comes in contact with H₂O₂, achemiluminescent reaction occurs. The effect of thiourea on this Luminolreaction was measured on the following reactions assembled in amicrotiter plate:

50 μl Luminol solution⁶

45 μl 0.0015% or 0.00015% H₂O₂

5 μl 0.5 M thiourea (25 mM final conc.) or water as control

⁶ Luminol solution per 100 ml:

0.4 gm sodium carbonate

0.02 gm luminol

2.4 gm sodium bicarbonate

0.05 gm ammonium carbonate

0.04 gm copper (II) sulfate pentahydrate

distilled water to 100 ml

pH 9.0

The resulting luminescence was immediately measured using a Dynex MLXMicrotiterplate luminometer or a Wallac 1450 MicroBeta Trilux plateluminometer (1 sec read per well). The results are listed in Table 3.The data demonstrate that thiourea acts on both CDP-Star® and Luminolchemiluminescence reactions to decrease autoluminescence.

TABLE 3 mM thiourea Fold decrease Substrate conc. (final) Formatluminescence CDP-Star 250 D1 50%    40 CDP-Star 125 D1 75%     6CDP-Star 25 D1 95%     2 CDP-Star 10 D2     1.6* Luminol 25 D3 150,0000.0015% H2O2 Luminol 25 D3   3840 0.00015% H2O2 *A parallel reaction wasconducted containing Alkaline Phospatase (AP) to monitor the effect ofthiourea on CDP-Star ® stability. The reaction containing AP alsodecreased the luminescence output but at a lesser magnitude than that ofthe CDP-Star ® alone. The AP reaction decreased 1.4 fold in the presenceof thiourea.

EXAMPLE 4 Effect of pH on Coelenterazine Derived Autoluminescence

The following experiment was performed to determine the ability ofthiourea to reduce autoluminescence at various pH values.

Matthew's buffer was made at a 2× concentration (standard formulation asdescribed in Example 1) and divided in several aliquots. Aliquots wereadjusted from pH 4 to pH 9 in one pH unit increments. For each pH,versions of the buffer were made with or without 1% Tergitol NP-9/1%antifoaming agent (referred to as “detergent”) and with or without 3 mMthiourea. Coelenterazine was added to a concentration of 180 mM for theversion with detergent, and to a concentration of 60 mM for the versionwithout detergent. The buffers were then brought to a final 1×concentration with water.

A 150 μl aliquot of each reaction was dispensed in triplicate intomicrotiter plate wells and the plate was incubated at 22° C. After 5minutes the luminescence was measured using a plate luminometer aspreviously described (1 sec per well) and the results are shown in thefollowing tables:

Matthew's Buffer with Detergent pH 4 pH 5 pH 6 pH 7 pH 8 pH 9Luminescence 1.45 3.78 56 137.5 224.7 361.1 without thiourea (relativelight units) Luminescence .0375 .04625 .21 .71 1.62 1.77 with 3 mMthiourea (relative light units) Fold Reduction 38.3 81.7 266.8 193.6138.4 204.4

Matthew's Buffer without Detergent pH 4 pH 5 pH 6 pH 7 pH 8 pH 9Luminescence 0.0925 0.2 0.78 3.63 39.62 56.36 without thiourea (relativelight units) Luminescence 0.025 0.024 0.063 0.259 1.52 1.785 with 3 mMthiourea (relative light units) Fold Reduction 3.7 8.4 12.4 14 26.1 31.6

These results demonstrate that increasing the pH of the buffer increasescoelenterazine auto luminescence. The addition of 3 mm thioureaeffectively decreases autoluminescence even at high pHs.

EXAMPLE 5 Reduction of Auto Luminescence in Whole Cell Assay

To determine the effect of organic compounds on cell viability and todetermine the ability of such compounds to reduce autoluminescence inthe presence of living cells, the following experiment was performed.Human embryonic kidney cells (293, ATCC, Rockville, Md.) were used togenerate a cell line that stably expresses the firefly luciferase (Luc+)gene. This stable cell line was made using the pCl-neo vector (PromegaCorporation, Madison Wis., USA) and inserting the Luc+ gene between theXba I and Sal I sites. The stable cell line was prepared using standardmethods and the transformed cells were grown in wells of a microtiterplate in the presence of DMEM medium containing 10% FBS and 0.15 mg/mlG418. For experimental purposes, duplicate plates of cells were preparedusing 96 well microtiter plates. One plate was used to examineviability, and the other plate was used to examine the effect onautoluminescence.

To examine the effect of the organic compounds in cell viability, themedia was removed from the cells and replaced with media containing thesubstrate for firefly luciferase, beetle luciferin, and the organiccompounds at various concentrations. Passive diffusion of luciferinacross the cell membrane together with the ATP oxygen and luciferaseenzyme already contained within the cell, results in light production.Whereas, in compromised or damaged cells, intracellular ATPconcentration is rapidly depleted, decreasing the firefly luminescence.The level of luminescence was compared to controls containing onlyluciferin to identify the effect of the compounds, if any, on lightoutput as an indicator of cell viability.

To determine the ability of the organic compounds to reduceautoluminescence, the media was removed from the cells and replaced withmedia containing the compounds at various concentrations andcoelenterazine. Since there is no Renilla luciferase enzyme beingexpressed in these cells, the only luminescence observed isautoluminescence. The level of autoluminescence was compared to controlscontaining only coelenterazine to identify the effect of the compoundson reducing autoluminescence.

Half of the microtiter plate contained no cells. To these wells, thesame reagents were added as to the cell counterpart to measurecell-independent luminescence (i.e. background luminescence). Thereagents (media with luciferin or coelenterazine, with or withoutcompounds at various concentrations) were prepared as follows:

a) Reagent to Examine Firefly Luminescence (Cell Viability)

Luciferin substrate available from Promega Corporation, Madison Wis.,USA was initially prepared in 10 mM sodium phosphate buffer, pH 7.4 as a100 mM stock. This luciferin stock was used to make DMEM solutioncontaining a final concentration of 2 mM luciferin.

Thiourea and 1-allyl-3-(2-hydroxyethyl)-2-thiourea were dissolveddirectly in this solution (DMEM/luciferin) to a final concentration of30 mM. These were subsequently diluted to contain final compoundconcentrations of 10 mM and 1 mM.

Another compound 6-aza-thio-thymidine was dissolved in DMSO as a 750 mMstock. The 6-aza-thio-thymidine was subsequently added to theDMEM/luciferin reagent at a final concentration of 30 mM, 10 mM, and 1mM, while maintaining a final DMSO concentration of 4%. A DMEM/luciferinreagent was used as the control with which to compare the effect of thecompound and was also made to contain a final DMSO concentration of 4%.

Since it is believed that DMSO may help organic molecules permeat cellmembranes, an additional control was included that consisted of thiourea(10 mM) reconstituted in DMSO (4%).

b) Reagent to Examine Reduction in Autoluminescence

Coelenterazine substrate was initially dissolved in Stop & Glo® ReagentSolvent (both available from Promega Corporation, Madison Wis., USA) asa 30 mM stock. This coelenterazine stock was used to make DMEM mediacontaining 0.6 mM coelenterazine as the final concentration.DMEM/coelenterazine reagents were made in a similar fashion as theDMEM/luciferin reagents described in a).

The cell culture medium was removed from the cells and replaced withmedium containing substrate (+/−compound) and luminescence was measuredimmediately. All luminescent values obtained from wells containing cellswere background subtracted using the corresponding luminescent valuesfrom those wells that did not contain cells. Fold reduction inautoluminescence was calculated by dividing the background-subtractedautoluminescence in minus compound controls by the background-subtractedautoluminescence containing the compounds.

Results for representative compounds are shown in the following

TABLE Effects on firefly Fold Decrease Concen- Additives lumin- in Auto-Compound tration to Media? escence luminescence Thiourea 30 mM No Yes,40 decreases Thiourea 10 mM No No 16 Thiourea  1 mM No No 3.4 Thiourea10 mM 4% DMSO No 12 1-Allyl-3-(2- 30 mM No Yes, 35 hydroxyethyl)-decreases 2-thiourea 1-Allyl-3-(2- 10 mM No Yes, 12 hydroxyethyl)-decreases 2-thiourea 1-Allyl-3-(2- 1 mM No No 3.5 hydroxyethyl)-2-thiourea 6-aza-thio- 30 mM 4% DMSO Yes, 525 thymidine decreases6-aza-thio- 10 mM 4% DMSO No 23 thymidine 6-aza-thio-  1 mM 4% DMSO No 3thymidine

These results demonstrate that these compounds can be used to reduceautoluminescence in luminescent assays employing whole cells withoutsignificantly decreasing cell viability.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications can be made while remainingwithin the spirit and scope of the invention.

1. A method for increasing the sensitivity of a bio-luminescent assaycomprising carrying out the assay in the presence of an organic compoundthat reduces luminescence that is not dependent on the presence of ananalyte by at least about 10 fold, and that reduces luminescence that isdependent on the presence of an analyte by less than about 7 fold,wherein the organic compound has at least one carbon-sulfur single bond(C—S), carbon-sulfur double bond (C═S), or carbon-selenium double bond(C═Se).
 2. The method of claim 1 wherein the luminescence that isdependent on the presence of an analyte is reduced by less than about 5fold.
 3. The method of claim 1 wherein the luminescence that isdependent on the presence of an analyte is reduced by less than about 2fold, remains the same, or is increased.
 4. The method of claim 1wherein the luminescence that is dependent on the presence of an analyteis maintained or increases.
 5. The method of claim 1 wherein theluminescence that is not dependent on the presence of an analyte ischemi-luminescence that does not result from a bio-luminescent reaction.6. The method of claim 1 wherein the luminescence that is dependent onthe presence of an analyte comprises luminescence generated within aliving cell.
 7. The method of claim 1 wherein luminescence that is notdependent on the presence of an analyte comprises luminescence generatedby a chemical reaction of coelenterazine or a functional analog thereof.8. A method for increasing the sensitivity of a luminescent assaycomprising carrying out the assay in the presence of an organic compoundthat reduces luminescence generated by luminogenic molecules not boundto an enzyme by at least about 10 fold, and that reduces theluminescence generated by luminogenic molecules bound to an enzyme byless than about 7 fold, wherein the organic compound has at least onecarbon-sulfur single bond (C—S), carbon-sulfur double bond (C═S), orcarbon-selenium double bond (C═Se).
 9. The method of claim 8 wherein theluminescence generated by luminogenic molecules bound to an enzyme isreduced by less than about 5 fold.
 10. The method of claim 8 wherein theluminescence generated by luminogenic molecules bound to an enzyme isreduced by less than about 2 fold, remains the same, or is increased.11. The method of claim 8 wherein the luminescence generated byluminogenic molecules bound to an enzyme is maintained or increases. 12.The method of claim 8 wherein the luminescence generated by luminogenicmolecules bound to an enzyme comprises luminescence generated within aliving cell.
 13. The method of claim 8 wherein the luminescencegenerated by luminogenic molecules not bound to an enzyme comprisesluminescence generated by a chemical reaction of coelenterazine or afunctional analog thereof.
 14. A method for increasing the sensitivityof a bio-luminescent assay comprising carrying out the assay in thepresence of an organic compound that reduces autoluminescence by atleast about 10 fold, and that reduces luminescence that is dependent onthe presence of an analyte by less than about 7 fold, wherein theorganic compound has at least one carbon-sulfur single bond (C—S),carbon-sulfur double bond (C═S), or carbon-selenium double bond (C═Se).15. The method of any one of claims 1–14 wherein the luminescent assayemploys a luciferase, aequorin, or obelin enzyme.
 16. The method of anyone of claims 1–14 wherein the luminescent assay employs fireflyluciferase.
 17. The method of any one of claims 1–14 wherein theluminescent assay employs Renilla luciferase.
 18. The method of any oneof claims 1–14 wherein the luminescent assay employs Cypridinaluciferase.
 19. The method of any one of claims 1–14 wherein the organiccompound is present in a concentration of at least 0.1 μM.
 20. Themethod of any one of claims 1–14 wherein the organic compound is presentin a concentration of at least 0.1 mM.
 21. The method of any one ofclaims 1–14 wherein the organic compound is present in a concentrationof from about 0.1 μM to about 500 mM.
 22. The method of any one ofclaims 1–14 wherein the organic compound is present in a concentrationof from about 100 μM to about 100 mM.
 23. The method of any one ofclaims 1–14 wherein the organic compound is present in a concentrationof from about 10 mM to about 100 mM.
 24. The method of any one of claims1–14 wherein the assay is performed in the presence of whole cells. 25.The method of any one of claims 1–14 wherein the assay is carried out ina solvent comprising at least about 10% water by weight.
 26. The methodof any one of claims 1–14 wherein the assay is carried out in a solventcomprising at least about 25% water by weight.
 27. The method of claim14 wherein the luminescence that is dependent on the presence of ananalyte is reduced by less than about 5 fold.
 28. The method of claim 14wherein the luminescence that is dependent on the presence of an analyteis reduced by less than about 2 fold, remains the same, or is increased.29. The method of claim 14 wherein the luminescence that is dependent onthe presence of an analyte is maintained or increases.
 30. The method ofclaim 14 wherein the luminescence that is dependent on the presence ofan analyte comprises luminescence generated within a living cell. 31.The method of claim 14 wherein the auto luminescence comprisesluminescence generated by a chemical reaction of coelenterazine or afunctional analog thereof.
 32. An assay kit comprising packagingmaterial containing 1) a luminogenic substrate of a luminescent enzyme,or a luminogenic enzyme; and 2) an organic compound in an amount forreducing luminescence that is not dependent on the presence of ananalyte by at least about 10 fold, and for reducing luminescence that isdependent on the presence of an analyte by less than about 7 fold,wherein the organic compound has at least one carbon-sulfur single bond(C—S), carbon-sulfur double bond (C═S), or carbon-selenium double bond(C═Se).
 33. The assay kit of claim 32 wherein the luminescence that isdependent on the presence of an analyte is maintained or increases. 34.The assay kit of claim 32 wherein the luminescence that is dependent onthe presence of an analyte comprises luminescence generated within aliving cell.
 35. The kit of claim 32, further comprising instructionsfor use of the organic compound in an amount for reducing luminescencethat is not dependent on the presence of an analyte by at least about 10fold, and for reducing luminescence that is dependent on the presence ofan analyte by less than about 7 fold.
 36. An assay kit comprisingpackaging material containing 1) a luminogenic substrate of aluminescent enzyme, or a luminogenic enzyme; and 2) an organic compoundfor reducing luminescence generated by luminogenic molecules not boundto an enzyme by at least about 10 fold, and for reducing luminescencegenerated by luminogenic molecules bound to an enzyme by less than about7 fold, wherein the organic compound has at least one carbon-sulfursingle bond (C—S), carbon-sulfur double bond (C═S), or carbon-seleniumdouble bond (C═Se).
 37. The assay kit of claim 36 wherein theluminescence generated by luminogenic molecules bound to an enzyme ismaintained or increases.
 38. The assay kit of claim 36 wherein theluminescence generated by luminogenic molecules bound to an enzymecomprises luminescence generated within a living cell.
 39. The kit ofclaim 36, further comprising instructions for use of the organiccompound in an amount for reducing luminescence generated by luminogenicmolecules not bound to an enzyme by at least about 10 fold, and forreducing luminescence generated by luminogenic molecules bound to anenzyme by less than about 7 fold.
 40. An assay kit comprising 1) aluminogenic substrate of a luminescent enzyme, or a luminogenic enzyme;and 2) an organic compound in an amount for reducing autoluminescence byat least about 10 fold, and for reducing luminescence that is dependenton the presence of an analyte by less than about 7 fold, wherein theorganic compound has at least one carbon-sulfur single bond (C—S),carbon-sulfur double bond (C═S), or carbon-selenium double bond (C═Se).41. The kit of any one of claims 32–40 wherein the enzyme substrate andthe compound are each contained in a separate container.
 42. The kit ofany one of claims 32–40 wherein the enzyme substrate and the compoundare contained in a single container.
 43. The kit of any one of claims32–40 further comprising a buffer solution suitable for use in aluminescent assay.
 44. The kit of claim 43 wherein the enzyme substrateand the buffer solution are contained in a single container.
 45. The kitof claim 43 wherein the compound and the buffer solution are containedin a single container.
 46. The kit of any one of claims 32–40 furthercomprising a substrate for a second luminescent enzyme.
 47. The kit ofany one of claims 32–40 further comprising a quenching agent for aluminescent enzyme reaction.
 48. The kit of any one of claims 32–40wherein the substrate is a substrate for firefly luciferase or asubstrate for Renilla luciferase.
 49. The kit of any one of claims 32–40further comprising ATP.
 50. The kit of any one of claims 32–40 thatcomprises both a luminogenic substrate of a luminescent enzyme, and aluminogenic enzyme.
 51. The assay kit of claim 40 wherein theluminescence that is dependent on the presence of an analyte ismaintained or increases.
 52. The assay kit of claim 40 wherein theluminescence that is dependent on the presence of an analyte comprisesluminescence generated within a living cell.
 53. The kit of claim 40,further comprising instructions for use of the organic compound in anamount for reducing autoluminescence by at least about 10 fold, and forreducing luminescence that is dependent on the presence of an analyte byless than about 7 fold.
 54. A method for increasing the sensitivity of abio-luminescent assay comprising carrying out the assay in the presenceof an organic compound that reduces the luminescence that does notresult from a bio-luminescent reaction by at least about 10 fold, andthat reduces luminescence that does result from a bio-luminescentreaction by less than about 7 fold, wherein the organic compound has atleast one carbon-sulfur single bond (C—S), carbon-sulfur double bond(C═S), or carbon-selenium double bond (C═Se).
 55. The method of claim 54wherein the luminescence that results from a bio-luminescent reaction ismaintained or increases.
 56. The method of claim 54 wherein theluminescence that does not result from a bio-luminescent reactioncomprises luminescence generated within a living cell.
 57. The method ofclaim 54 wherein the luminescence that does not result from abio-luminescent reaction comprises luminescence generated by a chemicalreaction of coelenterazine or a functional analog thereof.
 58. Themethod of any one of claims 1, 8, 14, and 54 wherein the organiccompound comprises a sulfur atom or a selenium atom.
 59. The method ofany one of claims 1, 8, 14, and 54 wherein the organic compound containsa carbon-sulfur double bond (C═S).
 60. The method of any one of claims1, 8, 14, and 54 wherein the organic compound contains a carbon-seleniumdouble bond (C═Se).
 61. The method of any one of claims 1, 8, 14, and 54wherein the organic compound is a compound of formula (I):

wherein X is S or Se; R₁ and R₂ are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₂–C₂₀)alkenyl,(C₂–C₂₀)alkynyl, aryl, heteroaryl, or NR_(a)R_(b); or R₁ and R₂ togetherwith the carbon to which they are attached form a 5, 6, 7, or 8 memberedsaturated or unsaturated ring comprising carbon and optionallycomprising 1, 2, or 3 heteroatoms selected from oxy (—O—), thio (—S—),or nitrogen (—NR_(c))—, wherein said ring is optionally substituted with1, 2, or 3 halo, hydroxy, oxo, thioxo, carboxy, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl; and R_(a), R_(b) and R_(c) are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkynyl, aryl, heteroaryl; wherein any(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₂–C₂₀)alkenyl(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, or (C₂–C₂₀)alkynyl of R₁, R₂,R_(a), R_(b), and R_(c) is optionally substituted with one or more halo,hydroxy, mercapto, oxo, thioxo, carboxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; and wherein any aryl orheteroaryl is optionally substituted with one or more halo, hydroxy,mercapto, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl;or a salt thereof.
 62. The method of any one of claims 1, 8, 14, and 54wherein the organic compound is a compound of formula R₃SH wherein R₃ is(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,aryl, or heteroaryl; wherein any (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl,(C₂–C₂₀)alkenyl, or (C₂–C₂₀)alkynyl of R₃ is optionally substituted withone or more halo, hydroxy, mercapto oxo, thioxo, carboxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, orNR_(d)R_(e); wherein R_(d) and R_(e) are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; andwherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, mercapto, hydroxy, oxo, carboxy, cyano,nitro, trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl; or a salt thereof.63. The method of any one of claims 1, 8, 14, and 54 wherein the organiccompound is a compound of formula R₄NCS wherein: R₄ is (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl; wherein any (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl,(C₂–C₂₀)alkenyl, or (C₂–C₂₀)alkynyl of R₄ is optionally substituted withone or more halo, hydroxy, mercapto, oxo, thioxo, carboxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, orNR_(f)R_(g); wherein R_(f) and R_(g) are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; andwherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, mercapto, hydroxy, oxo, carboxy, cyano,nitro, trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl; or a salt thereof.64. The method of any one of claims 1, 8, 14, and 54 wherein the organiccompound is a compound of formula R₅—X—R₆ wherein: X is —S— or —Se—; R₅is (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,aryl, or heteroaryl; and R₆ is hydrogen, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl; or R₅ and R₆ together with X form a heteroaryl; wherein any(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, or (C₂–C₂₀)alkynyl ofR₅ or R₆ is optionally substituted with one or more halo, hydroxy,mercapto, oxo, thioxo, carboxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, or NR_(k)R_(m); wherein R_(k)and R_(m) are each independently hydrogen, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; and wherein any aryl orheteroaryl is optionally substituted with one or more halo, mercapto,hydroxy, oxo, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl;or a salt thereof.
 65. The method of any one of claims 1, 8, 14, and 54wherein the organic compound is a compound of any one of formulae 1–11

.
 66. The method of any one of claims 1, 8, 14, and 54 wherein theorganic compound is a compound of formulae 1, 8, 15, or 17 as shown

.
 67. The method of any one of claim 1, 8, 14, or 54, wherein theorganic compound is not a polypeptide or protein comprising one or moremercapto (C—SH) groups.
 68. The method of any one of claim 1, 8, 14, or54, wherein the organic compound does not comprise one or more mercapto(C—SH) groups.
 69. An assay kit comprising packaging materialcontaining 1) a luminogenic substrate of an enzyme, or a luminogenicenzyme; and 2) an organic compound for reducing luminescence that doesnot result from a bio-luminescent reaction by at least about 10 fold,and that reduces luminescence does result from a bio-luminescentreaction by less than about 7 fold, wherein the organic compound has atleast one carbon-sulfur single bond (C—S), carbon-sulfur double bond(C═S), or carbon-selenium double bond (C═Se).
 70. The assay kit of claim69 wherein the luminescence that results from a bio-luminescent reactionis maintained or increases.
 71. The assay kit of claim 69 wherein theluminescence that does not result from a bio-luminescent reactioncomprises luminescence generated within a living cell.
 72. The kit ofclaim 69, further comprising instructions for use of the organiccompound in an amount for reducing luminescence that does not resultfrom a bio-luminescent reaction by at least about 10 fold, and forreducing luminescence that does result from a bio-luminescent reactionby less than about 7 fold.
 73. The kit of any one of claims 32, 36, 40,and 69 wherein the organic compound comprises a sulfur atom or aselenium atom.
 74. The kit of any one of claims 32, 36, 40, and 69wherein the organic compound contains a carbon-sulfur double bond (C═S).75. The kit of any one of claims 32, 36, 40, and 69 wherein the organiccompound contains a carbon-selenium double bond (C═Se).
 76. The kit ofany one of claims 32, 36, 40, and 69 wherein the organic compound is acompound of formula (I):

wherein X is S or Se; R₁ and R₂ are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₂–C₂₀)alkenyl,(C₂–C₂₀)alkynyl, aryl, heteroaryl, or NR_(a)R_(b); or R₁ and R₂ togetherwith the carbon to which they are attached form a 5, 6, 7, or 8 memberedsaturated or unsaturated ring comprising carbon and optionallycomprising 1, 2, or 3 heteroatoms selected from oxy (—O—), thio (—S—),or nitrogen (—NR_(c))—, wherein said ring is optionally substituted with1, 2, or 3 halo, hydroxy, oxo, thioxo, carboxy, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl; and R_(a), R_(b) and R_(c) are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, (C₂–C₂₀)alkynyl, aryl, heteroaryl; wherein any(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₁–C₂₀)alkoxy, (C₂–C₂₀)alkenyl(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, or (C₂–C₂₀)alkynyl of R₁, R₂,R_(a), R_(b), and R_(c) is optionally substituted with one or more halo,hydroxy, mercapto, oxo, thioxo, carboxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; and wherein any aryl orheteroaryl is optionally substituted with one or more halo, hydroxy,mercapto, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl;or a salt thereof.
 77. The kit of any one of claims 32, 36, 40, and 69wherein the organic compound is a compound of formula R₃SH wherein R₃ is(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,aryl, or heteroaryl; wherein any (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl,(C₂–C₂₀)alkenyl, or (C₂–C₂₀)alkynyl of R₃ is optionally substituted withone or more halo, hydroxy, mercapto, oxo, thioxo, carboxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, orNR_(d)R_(e); wherein R_(d) and R_(e) are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; andwherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, mercapto, hydroxy, oxo, carboxy, cyano,nitro, trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl; or a salt thereof.78. The kit of any one of claims 32, 36, 40, and 69 wherein the organiccompound is a compound of formula R₄NCS wherein: R₄ is (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl; wherein any (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl,(C₂–C₂₀)alkenyl, or (C₂–C₂₀)alkynyl of R₃ is optionally substituted withone or more halo, hydroxy, mercapto oxo, thioxo, carboxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, orNR_(f)R_(g); wherein R_(f) and R_(g) are each independently hydrogen,(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkoxycarbonyl aryl, or heteroaryl; andwherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, mercapto, hydroxy, oxo, carboxy, cyano,nitro, trifluoromethyl, trifluoromethoxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl; or a salt thereof.79. The kit of any one of claims 32, 36, 40, and 69 wherein the organiccompound is a compound of formula R₅—X—R₆ wherein: X is —S— or —Se—; R₅is (C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl,aryl, or heteroaryl; and R₆ is hydrogen, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, aryl, orheteroaryl; or R₅ and R₆ together with X form a heteroaryl; wherein any(C₁–C₂₀)alkyl, (C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, or (C₂–C₂₀)alkynyl ofR₅ or R₆ is optionally substituted with one or more halo, hydroxy,mercapto, oxo, thioxo, carboxy, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, heteroaryl, or NR_(k)R_(m); wherein R_(k)and R_(m) are each independently hydrogen, (C₁–C₂₀)alkyl,(C₃–C₈)cycloalkyl, (C₂–C₂₀)alkenyl, (C₂–C₂₀)alkynyl, (C₁–C₂₀)alkanoyl,(C₁–C₂₀)alkoxycarbonyl, aryl, or heteroaryl; and wherein any aryl orheteroaryl is optionally substituted with one or more halo, mercapto,hydroxy, oxo, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy,(C₁–C₂₀)alkanoyl, (C₁–C₂₀)alkanoyloxy, sulfo or (C₁–C₂₀)alkoxycarbonyl;or a salt thereof.
 80. The kit of any one of claims 32, 36, 40, and 69wherein the organic compound is a compound of any one of formulae 1–11

.
 81. The kit of any one of claims 32, 36, 40, and 69 wherein theorganic compound is a compound of formula 1, 2, 4, or 6

.
 82. The kit of any one of claim 32, 36, 40, or 69, wherein the organiccompound is not a polypeptide or protein comprising one or more mercapto(C—SH) groups.
 83. The kit of any one of claim 32, 36, 40, or 69,wherein the organic compound does not comprise one or more mercapto(C—SH) groups.